Rapid advances with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have transformed our ability to make targeted gene knockouts in zebrafish. Tools and techniques for making ZFN- or TALEN-mediated knockouts are becoming well refined and broadly distributed. Efforts supported by this grant over the previous three years have resulted in the development of publicly available protocols, reagents, and software for engineering ZFNs and TALENs and have generated zinc finger arrays and TALENs for a large set of endogenous zebrafish genes. As a result, today almost any zebrafish investigator can knock out their genes of interest using these resources. Despite the importance of gene knockouts for understanding gene function, null alleles represent only one tool for biological research in zebrafish. To understand gene function completely, methods for controlling gene expression both spatially and temporally would provide powerful tools. Engineered zinc finger and transcription activator-like effector repeat DNA-binding domains have the potential to contribute to all of these areas and to thereby further enhance the utility of the zebrafish as an experimental model. In this renewal application, we propose to continue our highly productive collaborative efforts and to move beyond routine gene knockouts to explore additional novel applications for engineered DNA-binding domains in the zebrafish. During the project period, several novel applications for TALEs will be developed, including the creation of artificial transcription factors, the construction of a set of TALENs tha will enable disruption of all zebrafish microRNAs, and the generation of several different types of conditional alleles. The specific aims are: Aim 1: To develop TALE-based transcription factors for controlling endogenous gene expression in zebrafish. Aim 2: To generate a comprehensive set of TALENs targeted to all zebrafish microRNAs (miRNAs). Aim 3: To develop novel tools that enable conditional alteration of gene structure and expression. By completing these aims, we will advance the field of genome editing and substantially enhance the utility of the zebrafish as an experimental model. Consistent with our strong and well-established commitment to open-source science, we will continue to make all of the reagents, protocols, and software needed to practice the technologies developed in this proposal publicly available to academic researchers. We believe that the team of researchers for this proposal has a strong track record of productivity working together and of generating tools and reagents that will be broadly useful to the community.